Observations on Red Mark Syndrome in juvenile rainbow trout farmed in RAS system

Red mark syndrome is a nonlethal skin disease, reported to affect farmed rainbow trout over 50 g worldwide (Metselaar et al., 2022). As reported by the results of the 25th Annual Workshop of the European Reference Laboratories for Fish Diseases (DTUAQUA, Denmark), RMS remains to date a major concern in rainbow trout farming. This syndrome has been studied extensively both in field and experimental cases, and diagnostic features have been established (Galeotti, Sarli, et al., 2021; Jorgensen et al., 2019; Oidtmann et al., 2013; VernerJeffreys et al., 2008). Slovenia and BosniaHerzegovina were the most recent European areas where RMS was reported to outbreak in intensive aquaculture farms (Galeotti, Manzano, et al., 2017; Galeotti, Volpatti, et al., 2021). Even though no aetiological agent has been proven through Koch's postulates, RMS has been consistently associated with the presence of Midichlorialike organisms which have been visualized in situ by transmission electron microscopy both in field and experimental cases (Cafiso et al., 2016; Galeotti, Ronza, & Beraldo, 2017; Metselaar et al., 2020; Orioles et al., 2022b). The outbreak under study occurred in a Slovenian rainbow trout recirculating aquaculture system (RAS) in March 2022 (water temperature 11– 12°C). Eggs came from a local domestic supplier. Fish were reared in concrete basins from 10 up to 150 g before being transferred to another farm to reach commercial size. Rainbow trout were fed a standard commercialpelleted diet. Water was taken from a local spring, and no other fish farms were supplied from the same water source either upstream or downstream. About 50% of fish were showing signs compatible with RMS as shown in Figure 1. No mortality was observed, and affected fish had normal food intake and growth rate.

Red mark syndrome is a non-lethal skin disease, reported to affect farmed rainbow trout over 50 g worldwide .
As reported by the results of the 25th Annual Workshop of the European Reference Laboratories for Fish Diseases (DTU-AQUA, Denmark), RMS remains to date a major concern in rainbow trout farming. This syndrome has been studied extensively both in field and experimental cases, and diagnostic features have been established (Galeotti, Sarli, et al., 2021;Jorgensen et al., 2019;Oidtmann et al., 2013;Verner-Jeffreys et al., 2008). Slovenia and Bosnia-Herzegovina were the most recent European areas where RMS was reported to outbreak in intensive aquaculture farms (Galeotti, Manzano, et al., 2017;. Even though no aetiological agent has been proven through Koch's postulates, RMS has been consistently associated with the presence of Midichlorialike organisms which have been visualized in situ by transmission electron microscopy both in field and experimental cases (Cafiso et al., 2016;Galeotti, Ronza, & Beraldo, 2017;Metselaar et al., 2020;Orioles et al., 2022b).
The outbreak under study occurred in a Slovenian rainbow trout recirculating aquaculture system (RAS) in March 2022 (water temperature 11-12°C). Eggs came from a local domestic supplier. Fish were reared in concrete basins from 10 up to 150 g before being transferred to another farm to reach commercial size. Rainbow trout were fed a standard commercial-pelleted diet.
Water was taken from a local spring, and no other fish farms were supplied from the same water source either upstream or downstream.
About 50% of fish were showing signs compatible with RMS as shown in Figure 1. No mortality was observed, and affected fish had normal food intake and growth rate.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. F I G U R E 1 Typical RMS macroscopic skin lesions. Gross skin lesions ranging from 1 to 2 cm were round to oval, reddish and covered by serous exudate associated with multifocal haemorrhages lesions were focal (8/10) to multifocal (2/10) placed on ventral flanks and abdomen. Desquamation and skin ulcers were present in 3 out of the 10 trout. Bar = 1 cm Ten symptomatic fish were sampled and killed with an overdose of MS222 for investigation purposes. Fish's weight ranged from 35 to 90 g and they were all in good body condition. Approximately, 1 ml of blood was collected from the caudal vein and put in a 1.5 ml sterile plain tube where it was allowed to clot to extract DNA. Samples of internal organs (gills, intestine, spleen, liver, kidney and heart) and samples from skin lesions and spleen were taken for histological and molecular biology analysis, respectively. Tissues obtained for histological examination from skin/muscle and internal organs were fixed in buffered formaldehyde and then processed by an automatic histoprocessor (TISBE, Diapath, Italy) to be embedded in paraffin (ParaplastPlus, Diapath). Serial 5 μm sections were stained with haematoxylin-eosin.
QIAmp DNA Mini extraction Kit (Qiagen, Hilden, Germany) was used on skin and spleen samples and on blood clots, using Tissue Lyser II equipment (Qiagen, Hilden, Germany). The concentration and purity of the extracted DNA were assessed using a 'NanoDrop One' spectrophotometer (Thermo Scientific, Waltham, MA, USA), and the DNA was then sent to the digital droplet PCR (ddPCR) analysis.
The ddPCR method was performed on the DNA extracted in accordance with recent literature ) and manufacturer's instructions (Bio-Rad, USA).
Gross skin lesions ranging from 1 to 2 cm were round to oval, reddish and covered by serous exudate associated with multifocal haemorrhages lesions were focal (8/10) to multifocal (2/10) placed on ventral flanks and abdomen. Desquamation and skin ulcers were present in 3 out of the 10 trout.
Necroscopy revealed splenomegaly in two fish. No abnormalities were detectable in the other organs. Cytology of skin and gills did not reveal the presence of parasites.
Histological examination of skin samples revealed a marked lymphocytic and macrophagic inflammatory infiltration involving all skin F I G U R E 2 (a-d): H&E sections. Histological features of RMS skin lesion. In early moderate cases (a), infiltration of the lymphocytic and macrophagic inflammatory cells involve predominantly dermis spongiosum and hypoderma; the same type of inflammatory infiltration involves all skin layers with progressive infiltration and thickening of dermis compactum, hypodermis and muscles in moderate and severe cases (b and c). Scale pocket (black stars) oedema, inflammatory infiltration and scale reabsorption by giant multinucleated cells (black arrows) are frequently observed (d) Histological examination of remaining organs did not reveal significant abnormalities.
Skin lesions were classified as moderate or severe as reported recently in the literature . Results of the molecular biology analysis are reported in Table 1.
Considering reported criteria for skin disorders in rainbow trout (Oidtmann et al., 2013) and the findings reported in this study, the skin lesions were consistent with RMS. This is the first report of RMS affecting rainbow trout in a RAS system and the evidence that RMS could affect rainbow trout under 50 g (smallest was

ACK N OWLED G EM ENT
Open Access Funding provided by Universita degli Studi di Udine within the CRUI-CARE Agreement.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request." cd_value_code="text